ISSN: 0215-6334 | e-ISSN: 1907-770X The Southeast Asian Journal of Tropical Biology Vol.
32 No.
2, 2025: 162 - 170
DOI: 10.
11598/btb.
Research Paper
THE EFFICACY OF BACTERIAL AND FUNGAL
ANTAGONIST SUSPENSIONS IN CONTROLLING
FOLIAR MILDEW DISEASE IN ZUCCHINI PLANTS
Yan Ramona1,2*.
Martin A.
Line3.
I Gusti Ayu Agung Septiari1.
Ida Bagus Gede Darmayasa1.
I Dewa Agung Panji Dwipayana1, and Kalidas Shetty4 Department of Biology.
Faculty of Mathematics and Natural Sciences.
Udayana University.
Badung 80361.
Bali.
Indonesia Integrated Laboratory for Biosciences and Biotechnology.
Udayana University.
Badung 80361.
Bali.
Indonesia School of Agricultural Sciences.
Faculty of Sciences and Engineering.
Tasmania University.
Dynnyrne TAS 7005.
Australia Department of Microbiology.
North Dakota State University.
Fargo.
ND 58105.
USA
ARTICLE HIGLIGHTS
ABSTRACT
A Diverse microbial antagonists can be used as alternatives to control foliar A Microbial agents offer zucchini protection against downy mildew causing microbes A Biocontrol agents effectively control mildew infection in zucchini plants A Diverse microbial antagonists have potential to control foliar disease in A New bio-based strategy supports sustainable crop disease management A Diverse microbial antagonists are promising for controlling mildew in Downy mildew is recognized as a major constraint in zucchini production, caused by obligate fungal-like pathogens that thrive under humid conditions.
In this study, the efficacy of selected bacterial (Lysobacter antibioticus Bali G.
Pseudomonas corrugata SAJ.
and fungal (Trichoderma sp.
Td.
antagonists was evaluated for the management of this foliar disease on zucchini plants as an alternative to chemical fungicides.
The efficacy of these bacterial and fungal antagonists against a suspected downy mildew pathogen was assessed on zucchini leaves in a glasshouse.
It was found that the antagonists provided 22 - 83% protection (P < 0.
against the pathogen two weeks after application.
However, the level of protection declined over time, with 46 - 60% of leaves infected five weeks after pathogen exposure, regardless of treatment.
The combination of Trichoderma sp.
Td22, the most effective agent, with either Lysobacter antibioticus Bali G.
Pseudomonas corrugata SAJ6, or both, was observed to reduce its overall effectiveness.
Survival of the biological agents on leaf surfaces was low, although prior research has indicated that survival may not be essential for sustained disease control.
Further investigation is required to determine the potential role of these agents in inducing systemic acquired resistance in crops such as grapes and poppies.
For commercial application, repeated treatments may be necessary to maintain disease management.
Notably, the protection provided by Trichoderma sp.
Td22 was found to be comparable to that of chemical treatments, representing a promising step toward more sustainable agricultural practices.
Article Information Received : 6 February 2025 Revised : 20 May 2025 Accepted : 1 August 2025 *Corresponding author, e-mail:
yan_ramona@unud.
Keywords: bacterial antagonists, biocontrol.
Lysobacter antibioticus.
Pseudomonas corrugata, zucchini disease control
INTRODUCTION
Downy mildew is a widespread and economically impactful plant disease that affects various crops, including cucumbers, grapes, cantaloupes, and other cucurbits, across different agricultural regions globally.
This disease is caused by obligate fungal-like pathogens, such as Plasmopara viticola (Heger et al.
Clippinger et al.
and Pseudoperonospora cubensis (Wallace et al.
Sun et al.
, which predominantly infect green plant tissues, especially the leaves.
Typical symptoms include irregular spots on the leaves, which can range from pale green to yellow or brown in color (Newark et al.
Purayannur et al.
Favorable conditions, including high humidity and moderate temperatures, facilitate rapid disease spread through wind-borne spores or rain splashes, often resulting in large-scale The efficacy of bacterial and fungal antagonist suspensions in controlling foliar mildew disease - Ramona et al.
In severe cases, downy mildew can lead to defoliation, stunted growth, poor fruit quality, and even total crop failure, making it a critical challenge for growers (Purayannur et al.
Marone Fassolo et al.
As global temperatures and weather patterns shift, the range and frequency of downy mildew outbreaks are projected to increase, emphasizing the need for integrated disease management strategies that combine resistant crop varieties, cultural practices, and sustainable control measures (Singh et al.
Globally, downy mildew significantly impacts food security and agricultural output, particularly in areas where susceptible crops are grown Plasmopara viticola for example, continues to challenge grape production in Europe.
North America, and Australia, leading to reduced harvests and increased dependency on fungicides (Koledenkova et al.
Similarly.
cubensis is a key limitation for cucurbit farming in Asia, the Americas, and parts of Africa, where recurrent outbreaks cause severe losses to commercial yields (Salcedo et al.
Downy mildew, a destructive disease affecting the foliage of many crops, has traditionally been managed using several strategies.
These include crop rotation, which interrupts the pathogenAos lifecycle by alternating host availability, and the deployment of resistant plant varieties that utilize genetic traits to fend off infections (Tyr et al.
Clippinger et al.
Efficient irrigation practices, such as drip systems and proper drainage, reduce moisture levels conducive to the diseaseAos growth (Tyr et al.
Clippinger et al.
Chemical fungicides remain a common solution, offering rapid disease suppression during critical periods (Toffolatti et al.
Clippinger et al.
However, their frequent use has raised concerns over environmental pollution, public health, and the evolution of fungicide-resistant pathogens (Ons et al.
Islam et al.
Current research underscores the value of integrated disease management (IDM) approaches that combine various practices to achieve effective and sustainable disease control.
Studies by Corkley et al.
and Wang et al.
demonstrated the advantages of integrating resistant crop varieties with targeted fungicide applications to manage disease outbreaks.
This dual approach, leveraging genetic resistance to reduce pathogen pressure and applying fungicides strategically to mitigate severe infections, minimizes reliance on chemicals while sustaining crop yields.
Such strategies align with contemporary goals of reducing the environmental impact of agriculture.
In growing concerns about fungicide overuse, biological control options are becoming increasingly popular within IDM frameworks (Ons et al.
Compost teas, liquid extracts produced through compost fermentation, represent one promising strategy (Coker & Ozores-Hampton 2.
These solutions are rich in beneficial microorganisms and bioactive compounds that combat pathogens and enhance plant immunity (Ramyrez-Gottfried et al.
They offer a natural and sustainable alternative or complement to synthetic chemicals, integrating seamlessly into environmentally conscious agricultural practices.
Recent research also highlights the role of specific biocontrol agents, such as L.
corrugata, and Trichoderma spp.
in managing zucchini diseases such as downy mildew (Ayaz et antibioticus produces antibiotics that suppress Pseudoperonospora cubensis (Drenker et al.
, the pathogen responsible for downy mildew, while P.
corrugata inhibits spore germination and disease spread through antagonistic interactions (Ramona et al.
Meanwhile.
Trichoderma sp.
not only protect plants by colonizing root systems but also enhance systemic resistance, making plants more resilient to infections (Chakraborty et Combining traditional practices with these innovative biological tools provides a balanced way forward (Clippinger et al.
This integrated approach reduces harmful inputs while addressing key challenges in plant health management, promoting sustainable and resilient agricultural The effectiveness of compost teas in managing plant diseases arises primarily from the diversity of their microbial communities, which actively suppress pathogens while supporting plant health (Barghouth et al.
Beneficial microbes, such as Bacillus and Pseudomonas produce antimicrobial compounds that inhibit pathogen growth, while fungi like Trichoderma outcompete pathogens for space and nutrients and form protective root barriers (St.
Martin et al.
Some microorganisms in compost teas also trigger systemic resistance, equipping plants with enhanced defense mechanisms against diverse pathogens (Emannuel Oliveira Vieira et al.
BIOTROPIA Vol.
32 No.
2, 2025
Sarmah et al.
emphasized that enriching compost teas with targeted strains, including Trichoderma spp.
and Bacillus spp.
, boosts their efficacy against a broader spectrum of diseases.
This approach addresses common challenges like variability in compost tea effectiveness, which often results from differences in compost quality and brewing conditions.
Introducing wellcharacterized microbial strains ensures consistency and reliability, enhancing the utility of compost teas in agriculture.
Beyond their role in disease management, compost teas promote healthier plants by improving soil quality and nutrient availability (De Corato, 2020.
Ramyrez-Gottfried et al.
Their microorganisms facilitate the release of essential nutrients like nitrogen and phosphorus, boosting plant growth and vitality (Singh et al.
By combining disease suppression with nutrient enhancement, compost teas serve as a multifunctional tool that aligns with the goals of sustainable agriculture.
Leveraging these biological solutions offers a scalable and eco-friendly way to address modern agricultural challenges.
Based on the above rationale, this research focused on assessing the effectiveness of bacterial suspensions of Lysobacter antibioticus Bali G and Pseudomonas corrugata SAJ 6 in TSB, as well as a fungal spore suspension of Td22 in saline, for controlling downy mildew in zucchini plants.
The study aimed to offer alternatives to conventional Aucompost teasAy by using selective microbial antagonists as active agents for disease management in important crops, including, zucchini, grapes, and poppies.
MATERIALS AND METHODS
Bacterial and Fungal Antagonist Isolates Three antagonistic microorganisms, including Lysobacter antibioticus Bali G.
Pseudomonas corrugata SAJ6, and Trichoderma sp.
Td22 that effective against Sclerotinia minor in lettuce plants (Ramona et al.
, were evaluated in the current study for their potential to manage a foliar mildew disease, downy mildew, in zucchini.
The L.
antibioticus Bali G and P.
corrugata SAJ6 were obtained from lettuce farms in Bedugul.
Bali.
Indonesia, whereas the Trichoderma sp.
Td22 was obtained from Dr Dean Metcalf, a senior researcher at Department of Primary Industries.
Parks.
Water, and Environment (DPIWE) in Tasmania.
Australia.
Downy Mildew Isolate The pathogen analyzed in this research was obtained from diseased grape leaves sourced from the Horticultural Research Centre (HRC) at the University of Tasmania.
Australia.
The characteristic leaf damage initially suggested identification as downy mildew.
As obligate parasites, downy mildew pathogens necessitate the need for living therefore, the infected leaves were collected just prior to preparing the pathogen suspension in saline solution.
Preparation of Antagonist Suspensions The bacterial antagonists were grown in a medium containing 0.
5% .
trypticase soya broth (OXOID) at 25 AC for 48 hours without agitation, achieving a final concentration of roughly 10A cells/mL.
The Trichoderma sp.
Td22 spores were obtained from wood fiber waste (WFW) compost, which had previously supported Td22 cultivation during our previous lettuce or pyrethrum experiments.
To extract the spores, the Td22-grown WFW compost was agitated to release most spores in a saline solution at a 1 : 10 .
ratio for around 10 minutes before being The Trichoderma sp.
Td22 spore density obtained was 8.
42 A 0.
01 log10 cfu/mL .
verage of triplicates measurements with an Improved New Bauer Hemacytomete.
Preparation of the Pathogen Suspension Approximately 10 g of infected grape leaves were placed in 200 mL of sterile saline solution .
85% NaC.
and shaken thoroughly to release the pathogen from the leaves.
The mixture was then sieved with a piece of sterile cloth to remove the leaf debris, before being used in the trials.
density of 2.
7 x 107 propagules/mL were obtained following determination with an Improved New Bauer Hemacytometer.
Glasshouse Scale Experiments Zucchini seeds (AoBlackjackAo YatesA) were sown in 5 L pots containing a steam-sterilized standard potting mix.
After 14 days, the seedlingsAo leaves were sprayed with 2 mL of antagonist suspensions.
The study included various combinations of antagonists, such as L.
antibioticus P.
antibioticus Trichoderma sp.
Td22.
Trichoderma sp.
Td22, and a mixture of all three (L.
antibioticus P.
corrugata Trichoderma sp.
Td.
in equal proportions .
Three days following the antagonist application, a 2 mL suspension of The efficacy of bacterial and fungal antagonist suspensions in controlling foliar mildew disease - Ramona et al.
the pathogen was sprayed onto the leaves.
Each treatment consisted of five replicate pots, with each pot holding a single 3 weeks old plant or approximately 10 cm in height.
Control groups were either sprayed with only the pathogen or with a saline solution lacking both pathogens and The pots were maintained in a shaded house for eight weeks, with infection levels evaluated at two and five weeks post-pathogen application.
avoid cross-contamination, control pots (A0B.
were positioned separately from those exposed to the pathogen.
Infection severity on the leaves was assessed using a 0 - 5 scale, as described by Nakasaki et al.
, where 0 indicated no visible symptoms, 1 represented infection on O 20% of the leaf area, 2 on 21 - 40%, 3 on 41 - 60%, 4 on 61 - 80%, and 5 on 81 - 100%.
Establishment of the Antagonists on the Zucchini Leaves The experiment was completed six weeks after the pathogen inoculation, with efforts made to recover antagonists from randomly chosen healthy To assess the colonization of bacterial antagonists, 10 g of leaves from each pot were mixed with 90 mL of saline solution and homogenized for 3 - 5 minutes.
Colony-forming units .
were quantified using dilution plating on Trypticase Soya Agar (TSA), followed by incubation at 25 AC for 2 - 5 days.
The identities of the bacteria were verified by comparing colony characteristics on TSA with those of the original strains To evaluate the presence of Trichoderma sp.
Td22, 20 leaf plugs .
x3 m.
per treatment were aseptically collected and placed on pectin agar medium (MERCK) containing 60 AAg/mL These samples were incubated at 25 AC for 4 - 7 days to allow fungal growth.
Emerging fungal colonies were isolated and grown on the same medium to compare their morphology with a Trichoderma sp.
Td22 stock culture.
Observations were extended for one week to monitor conidial development for accurate identification.
Data Analysis The data obtained from this study were analyzed using analysis of variance (ANOVA), which was carried out with the help of Minitab software for Windows.
ANOVA enabled the assessment of any significant variations between the treatment groups.
To identify specific differences between group means, the least significant difference (LSD) test was employed following the ANOVA procedure.
The LSD test is a post-hoc statistical test that compares means to detect significant differences.
A significance threshold of P < 0.
05 was set to determine whether the differences observed were statistically meaningful, ensuring the results were valid and reliable.
RESULTS AND DISCUSSION
The efficacy of the selected antagonistic fungus and bacteria in preventing zucchini leaves from downy mildew infection, as evaluated by the percentage of infected leaves and the disease severity index is presented in Figure 1.
The use of antagonists, excluding treatment A2B1 .
lants treated with L.
antibioticus and the pathoge.
, significantly lowered disease incidence compared to the untreated-pathogen control (A0B.
two weeks post-infection (P < 0.
(Fig.
The most effective disease suppression was achieved with the fungal antagonist Trichoderma Td22 (A3B.
, providing an 83% reduction in disease .
alculated from disease incidenc.
compared to the control group at two-weeks postinoculation.
However, this effect declined and became statistically insignificant after five weeks.
A non-significant synergistic effect (P > 0.
was noted when L.
antibioticus and P.
corrugata were applied in combination .
reatment A4B.
, resulting in 57% disease protection .
alculated from disease Fig.
1A) relative to the untreatedpathogen control.
This was higher .
igher protectio.
than when each bacterial antagonist was applied individually (A1B1 or A2B1.
Fig.
When the Trichoderma sp.
Td22 was combined with the bacterial antagonists, its effectiveness diminished, possibly due to reduced levels of the primary biocidal compounds on leaf surfaces (Poromarto et al.
Additionally, the lack of synergy between the fungal and bacterial antagonists could be from antagonistic interactions, as dual-culture tests showed inhibition zones produced by both bacteria against Trichoderma sp.
Td22 (Fig.
contrast to our findings.
Poveda & Eugui .
suggested synergic effect when they were applied in combination in a sustainable agriculture system.
BIOTROPIA Vol.
32 No.
2, 2025
Figure 1 The effectiveness of Lysobacter antibioticus Bali G.
Pseudomonas corrugata SAJ6, and Trichoderma sp.
Td22 to prevent zucchini plants from foliar downy mildew infection in a glasshouse scale experiment Notes: A = percentage of infected leaves.
B = disease severity index.
the assessments were conducted at 2 and 5 weeks after infection.
treatments applied: A0B0 = control group .
o antagonist or pathogen applie.
A0B1 = control treatment .
athogen only applie.
A1B1 = plants treated with P.
corrugata and pathogen.
A2B1 = plants treated with L.
antibioticus and pathogen.
A3B1 = plants treated with Td22 and pathogen.
A4B1 = plants treated with a mixture of L.
corrugata, and pathogen.
A5B1 = plants treated with a mixture of L.
Td22, and pathogen.
A6B1 = plants treated with a mixture of P.
Td22, and pathogen.
A7B1 = plants treated with a mixture of all three antagonists and pathogen.
Each bar represents the mean of disease A standard error.
Figure 2 In vitro dual culture assays between L.
antibioticus Bali G and Trichoderma sp.
Td22 .
and between P.
corrugata SAJ 6 and Trichoderma sp.
Td22 .
on TSA plates after incubation at 25 AC for three days An interesting phenomenon was observed in this glasshouse trial where some plants in the control group .
o antagonist or pathogen applied or A0B.
showed disease symptom (Fig.
This could be due to cross contamination from the infected leaves nearby .
lants in pots treated with Water splash during irrigation or the blowing wind could be the main cause of this cross The protection shown by the antagonists against downy mildew was no longer significant (P > 0.
after five weeks.
At that time, 60% of the leaves in the untreated control group were infected.
The survival rate of the applied antagonists on the leaf surfaces was observed to be low.
For instance.
Trichoderma Td22 was recovered from only two out of 20 leaf samples taken from treatment A5B1 .
lants treated with L.
Trichoderma sp.
Td22, and the pathoge.
five weeks post-application, and it was The efficacy of bacterial and fungal antagonist suspensions in controlling foliar mildew disease - Ramona et al.
undetectable in samples from other treatments.
Likewise, the bacterial antagonists applied to the leaves were not found .
from any treatments after the five-week exposure.
Applying fungal and bacterial antagonists, either individually or in combination, showed potential for managing foliar diseases, such as downy mildew in zucchini plants.
Significant reductions in disease incidence (P < 0.
were observed two weeks after the pathogen was introduced (Fig.
These findings indicate the possibility of modifying compost to develop specialized Aucompost teasAy or cultivating specific biocontrol agents to manage foliar pathogens.
The application of Trichoderma sp.
Td22 yielded the most successful results in our study, particularly when the strain was cultivated in a mixture of wood fiber waste (WFW) compost and millet seed .
: 20 w/.
, as detailed in our previous research (Ramona et al.
The biocontrol agent was stored for approximately 10 months at around 20 AC before being applied in the current study.
efforts to further improve the effectiveness of both fungal and bacterial antagonists, recent research by Brost .
suggested that the addition of chelating agents and detergents may enhance the activity of these biocontrol agents.
By incorporating such additives, it may be possible to increase the stability, viability, and overall efficacy of microbial antagonists under various application conditions.
Previous studies have pointed out the compatibility challenges between bacterial and fungal antagonists, which can significantly influence the success of biocontrol strategies.
ElSharkawy et al.
for example, reported that the combination of T.
harzianum with P.
reduced the efficacy of the fungus in controlling Aphanomyces euteiches, a root rot pathogen in This finding highlights the importance of understanding microbial interactions, as incompatibility can undermine the individual effectiveness of biocontrol agents.
On the other hand, research by Amirthalingam et al.
and Ntakirutimana et al.
showed that using mixed cultures of antagonistic microorganisms often improved disease control.
These contrasting outcomes emphasize the need for comprehensive evaluations of compatibility when developing microbial formulations for disease management.
The broad-spectrum potential of Trichoderma strains, such as Td22, is particularly significant in this regard.
Its ability to combat various fungal pathogens has been consistently demonstrated in several studies, establishing it as a promising biocontrol agent (Ali et al.
Kumar et al.
In our current study.
Trichoderma sp.
Td22 achieved an 83% reduction in zucchini downy mildew during a two-week glasshouse trial, offering a level of protection comparable to chemical Such results validate its potential as a sustainable alternative to synthetic treatments.
However, achieving similar effectiveness in field conditions remains challenging, as environmental factors often necessitate frequent reapplication to maintain the agentAos activity and persistence.
Reapplication intervals for biocontrol agents align closely with those recommended chemical fungicides.
Jones et al.
for example, suggested reapplying fungicides every 10 - 14 days to sustain protection against downy This similarity underscores the practical challenges of deploying biological control agents while highlighting their potential integration into established disease management practices.
Optimizing formulations, improving the stability of biocontrol products, and addressing compatibility issues are essential for advancing the reliability and scalability of biological control solutions.
Mildew symptoms observed in the nil-pathogen control group (A0B.
after five weeks (Fig.
were likely caused by natural infection from spores originating in a nearby vineyard with known disease presence or accidental transfer through human activity.
Despite this unintended exposure, the infection levels remained minimal compared to those in inoculated plants, ensuring that the overall conclusions of the study were unaffected.
This minimal infection demonstrates the robustness of the experimental setup in isolating key variables under investigation.
The limited persistence of biocontrol agents on leaf surfaces was expected due to several environmental challenges.
Factors such as low moisture levels, ultraviolet (UV) radiation from sunlight, and the washing effect of overhead irrigation contributed to the reduced survival of these agents (Devi 2.
These environmental conditions are well-known to limit the effectiveness of biocontrol organisms in outdoor applications.
thorough review by Fedele et al.
discussed the impacts of environmental stressors, including humidity, temperature variations, and irrigation practices, on the survival and performance of biocontrol agents in field environments.
These BIOTROPIA Vol.
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findings emphasize the need for improved application techniques and protective formulations to enhance the stability and efficacy of biocontrol agents under field conditions.
CONCLUSION
The biological control agents evaluated in this study, with the exception of L.
antibioticus Bali G, demonstrated significant effectiveness (P < 0.
in protecting zucchini leaves from downy mildew during the first two weeks of the glasshouse trial.
These findings highlighted their potential for managing foliar diseases.
It is necessary to maintain protection beyond 14 days by reapplying the biocontrol agents.
The fungal antagonist Trichoderma sp.
Td22 was found to be incompatible with both L.
Bali G and P.
corrugata SAJ6.
In contrast, the combination of L.
antibioticus Bali G and P.
corrugata SAJ6 appeared to be compatible but did not significantly enhanced disease control when compared to P.
corrugata SAJ6 alone.
The survival of all three antagonists on zucchini leaf surfaces was notably very low.
ACKNOWLEDGMENT
The authors express their gratitude to the Ministry of Higher Education.
Research, and Technology of the Republic of Indonesia for partially funding this research.
Appreciation is also extended to Dr.
Dean Metcalf, the head of Integrated Laboratory for Biosciences and Biotechnology.
Udayana University, and the School of Agricultural Sciences.
Faculty of Sciences and Engineering.
Tasmania University for providing the Trichoderma isolate, necessary consumables, and equipment, respectively, used in this study.
Our acknowledgment should also go to Prof.
John Bowman for his assistance in the molecular identification of our bacterial isolates.
REFERENCES